This invention is directed to assay devices for determination of characteristics of samples, unitized housings, kits incorporating the assay devices, and methods of determining the characteristics of samples using the assay devices.
Among the many analytical systems used for detection or determination of analytes, particularly analytes of biological interest, are chromatographic assay systems. Among the analytes frequently assayed with such systems are:
(1) hormones, such as human chorionic gonadotropin (hCG), frequently assayed as a marker of human pregnancy, as well as luteinizing hormone (LH), follicle stimulating hormone (FSH), and thyroid stimulating hormone (TSH);
(2) antigens, particularly antigens specific to bacterial, viral, and protozoan pathogens, such as Streptococcus, hepatitis virus, and Giardia;
(3) antibodies, particularly antibodies induced as a result of infection with pathogens, such as antibodies to the bacterium Helicobacter pylory and to human immunodeficiency virus (HIV);
(4) other proteins, such as hemoglobin, frequently assayed in determinations of fecal occult blood, an early indicator of gastrointestinal disorders such as colon cancer;
(5) enzymes, such as aspartate aminotransferase, lactate dehydrogenase, alkaline phosphatase, and glutamate dehydrogenase, frequently assayed as indicators of physiological function and tissue damage;
(6) drugs, both therapeutic drugs, such as antibiotics, tranquilizers, and anticonvulsants, and illegal drugs of abuse, such as cocaine, heroin, amphetamines, and marijuana; and
(7) vitamins.
Such chromatographic systems are frequently used by physicians and medical technicians for rapid in-office diagnosis or therapeutic monitoring of a variety of conditions and disorders. They are also increasingly used by patients themselves for at-home monitoring of such conditions and disorders.
Among the most important of such systems are the xe2x80x9cthin-layerxe2x80x9d systems in which a solvent moves across a thin, flat, absorbent medium. Among the most important of tests that can be performed with such thin-layer systems are immunoassays, which depend on the specific interaction between an antigen or hapten and a corresponding antibody or other specific binding partner. The use of immunoassays as a means for testing for the presence or amount of clinically important molecules has been known for some time. As early as 1956, J. M. Singer reported the use of an immune-based latex agglutination test for detecting a factor associated with rheumatoid arthritis (J. M. Singer et al., Am. J. Med., 22:888-892 (1956)).
Among the chromatographic techniques used in conjunction with immunoassays is a procedure known as immunochromatography. In general, this technique uses a disclosing reagent or particle that has been linked to an antibody to the molecule to be assayed, forming a conjugate. This conjugate is then mixed with a specimen and, if the molecule to be assayed is present in the specimen, the disclosing reagent-linked antibodies bind to the molecule to be assayed, thereby giving an indication that the molecule to be assayed is present. The disclosing reagent or particle can be identifiable by color, magnetic properties, radioactivity, enzymatic activity, fluorescence, chemiluminescence, specific reactivity with another molecule, or another physical or chemical property. The specific reactions that are employed vary with the nature of the molecule being assayed and the sample to be tested.
Immunochromatographic assays fall into two principal categories: xe2x80x9csandwichxe2x80x9d and xe2x80x9ccompetitive,xe2x80x9d according to the nature of the antigen-antibody complex to be detected and the sequence of reactions required to produce that complex. In general, the sandwich immunochromatographic procedures call for mixing the sample that may contain the analyte to be assayed with antibodies to the analyte. These antibodies are mobile and typically are linked to a label or disclosing reagent, such as dyed latex, a colloidal metal sol, a nonmetallic colloidal sol, or a radioisotope. This mixture is then applied to a chromatographic medium containing a band or zone. This band or zone contains immobilized antibodies to the analyte of interest. The chromatographic medium is often in the form of a strip resembling a dipstick. When the complex of the molecule to be assayed and the labeled antibody reaches the zone of the immobilized antibodies on the chromatographic medium, binding occurs and the bound labeled antibodies are localized at the zone. This indicates the presence of the molecule to be assayed. This technique can be used to obtain quantitative or semi-quantitative results.
Examples of sandwich immunoassays performed on test strips are described by U.S. Pat. No. 4,168,146 to Grubb et al. and U.S. Pat. No. 4,366,241 to Tom et al., both of which are incorporated herein by this reference.
In a competitive immunoassay, typically, a labeled analyte or analogue is supplied, and a competitive reaction is set up between the unlabeled analyte in the sample and the labeled analyte or analogue for binding to an immobilized specific binding partner on the test strip. In general, competitive immunoassays are more suitable for assay of haptens, because they do not require the formation of a ternary sandwich complex.
Although useful, currently available chromatographic techniques using test strips have a number of drawbacks. Many samples, such as fecal samples, contain particulate matter that can clog the pores of the chromatographic medium, greatly hindering the immunochromatographic process. Other samples, such as blood, contain cells and colored components that make it difficult to read the results of the tests. Even if the sample does not create interference, it is frequently difficult with existing chromatographic test devices to apply the sample to the chromatographic medium so that the sample front moves uniformly through the chromatographic medium to ensure that the sample reaches the area where the binding is to occur in a uniform, straight-line manner.
Sample preparation and waste generation are responsible for other problems with currently available devices and techniques for immunochromatography. The increased prevalence of diseases spread by infected blood and blood fractions, such as AIDS and hepatitis, has exacerbated these problems. It is rarely possible to apply a sample (such as blood or feces) or a sampling device (such as a throat swab) directly to the chromatographic medium. Several extraction and pretreatment reactions are usually required before the sample can be applied to the chromatographic medium. These reactions are typically carried out by the physician or technician performing the test in several small vessels, such as test tubes or microfuge tubes, requiring the use of transfer devices such as pipettes. Each of these devices is then contaminated and must be disposed of using special precautions so that workers or people who might inadvertently come into contact with the waste do not become contaminated and subject to infection by infectious agents contained in the waste.
When blood samples are involved, there are additional considerations. Whole blood samples are most easily accommodated by the reverse flow format, i.e., one that performs bidirectional chromatography. The reverse-flow format has the advantage of allowing the test results to be read against the clear white or translucent chromatographic medium, eliminating the potential for obscuring weak test results. However, in a such a reverse-flow format, the amount of sample that is brought into contact with the capture line or detection zone is very limited. In a typical assay, approximately only 3-5 xcexcl of the applied sample is utilized, and hence assay sensitivity is xe2x80x9ccapture limited.xe2x80x9d This is especially true if the immunological reagents used are of low affinity or avidity for the performance of immunochromatographic assays. Therefore, it would be desirable to have an immunochromatographic assay device that could perform assays for analytes found in blood with a unidirectional assay format so that the sensitivity of the assay could be increased.
Additionally, such a device should be capable of receiving a possibly contaminated sample or a sample preparation device directly so as to eliminate the need for extraction vessels and transfer devices. Such a device, preferably in the form of a test strip, should also be capable of performing immunochromatographic assays on colored samples or samples containing particulates without interference and should be able to deliver the sample to the chromatographic medium uniformly and evenly to improve accuracy and precision of the test. This aspect of an improved assay device is particularly important in avoiding false negatives and false positives. In particular, an improved assay device should also be capable of performing a unidirectional assay on whole blood samples.
Another aspect in which present immunochromatographic test devices need improvement is in reducing the volume of sample required to achieve the threshold sensitivity of the analyte to be determined. This is particularly important if the sample is whole blood and the donor is an infant, patient suffering from blood loss or anemia pediatric patient, or geriatric patient, limiting the volume of blood that can be drawn. If multiple tests are to be performed, as is often the case, it is crucial that the minimum volume of blood be used for each test.
Another improvement in immunochromatographic assay test devices and formats is related to the use of monoclonal antibodies in assay test formats, particularly for assay of hCG and related hormones such as LH, FSH, and TSH. Although monoclonal antibodies can yield an increase in sensitivity and specificity for such assays, they can cause additional problems, particularly when murine monoclonal antibodies are used. If a plasma or serum sample is used, the human-anti-murine antibody (HAMA) response can cause interference in the assay when monoclonal antibodies are used. Therefore, there is a need to control and monitor the existence of such interference and provide an indication to the user that such interference does not exist in a particular assay.
Accordingly, there is a need for an improved immunochromatographic assay device that can perform unidirectional assays with blood samples and that can provide a high degree of sensitivity and reproducibility in a unidirectional immunochromatography format.
A chromatographic assay device according to the present invention meets these needs and provides a unidirectional immunoassay for an analyte in a blood sample while preventing interference from blood cells and colored components. The ability to perform a unidirectional immunoassay for an analyte in a blood sample provides improved sensitivity for detection of analytes such as hormones.
An embodiment of a chromatographic immunoassay device according to the present invention comprises:
(1) a first opposable component including:
(a) a sample application zone containing a matrix of porous material permeable to the liquid portion of blood but capable of trapping the cellular components of blood; and
(b) a chromatographic medium having first and second ends and including:
(i) a detection zone having immobilized thereon a first specific binding partner for the analyte; and
(ii) a conjugate zone having a labeled second specific binding partner for the analyte in a resolubilizable form; the sample application zone being in operable contact with the first end of the chromatographic medium and the conjugate zone being located closer to the first end of the chromatographic medium than the detection zone; and
(2) a second opposable component including:
(a) an applicator; and
(b) an absorber.
In this device, the first and second opposable components are brought into operable contact to cause the applicator to come into operable contact with the sample application zone to apply a wash liquid thereto and to cause the absorber to come into operable contact with the second end of the chromatographic medium.
Typically, the label is a colloidal particle label. Preferably, the colloidal particle label is a colloidal carbon label. Typically, the label is a visibly detectable label.
The analyte can be selected from the group consisting of human chorionic gonadotropin (hCG), luteinizing hormone (LH), follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH), corticotropin-releasing hormone (CRH), growth hormone-releasing hormone (GHRH), prolactin, human growth hormone (hGH), xcex2-lipotropin, corticotropin, xcex2-endorphin, calcitonin, parathormone, human placental lactogen (hPL), insulin, glucagon, pancreatic polypeptide (PP), secretin, cholecystokinin-pancreozyrnin, motilin, vasoactive intestinal peptide, gastric inhibitory peptide, and erythropoietin. Particularly significant analytes include human chorionic gonadotropin (hCG), luteinizing hormone (LH), follicle stimulating hormone (FSH), and thyroid stimulating hormone (TSH).
When the analyte is hCG, typically the first specific binding partner is a monoclonal antibody specific for the xcex1 subunit of hCG and the second specific binding partner is a monoclonal antibody specific for the xcex2 subunit of hCG and not cross-reactive with luteinizing hormone. Alternative arrangements of monoclonal antibodies can also be used, such as having the first specific binding partner being a monoclonal antibody specific for the xcex2 subunit of hCG and the second specific binding partner being specific for the cc subunit of hCG.
Preferably, the device further comprises a barrier to prevent fluid now between the second applicator and the sample application zone.
Preferably, the matrix contains a detergent and a chelating agent. The detergent can be a polyoxyethylene sorbitan monolaurate, a polyoxyethylene sorbitan monooleate, a polyoxyethylene sorbitan monopalmitate, a polyoxyethylene sorbitan monostearate, a polyoxyethylene sorbitan trioleate, a polyethylene glycol fatty alcohol ether, a polyoxyethylene fatty acid ester, or a polyoxyethylene ether. Most preferably, the detergent is a polyoxyethylene sorbitan monolaurate.
The chelating agent can be EDTA or EGTA. Preferably, the chelating agent is EDTA.
In the device, the chromatographic medium can further include a control zone separate from the detection zone. The control zone can include an analyte or an analyte analogue immobilized thereto. Alternatively, the control zone can include an immobilized third specific binding partner that does not bind the analyte but does specifically bind the labeled second specific binding partner.
Another aspect of the present invention is a test kit. The test kit comprises, packaged in separate containers:
(1) the chromatographic assay device described above;.and
(2) a wash liquid for application to the second applicator of the assay device.
Another aspect of the present invention is a method for detecting or determining an analyte in a test sample comprising the steps of:
(1) applying a test sample to the sample application zone of the chromatographic assay device described above;
(2) applying a wash liquid to the applicator of the chromatographic assay device;
(3) allowing the sample to migrate from the sample application zone through the chromatographic medium including the conjugate zone and the detection zone to resolubilize the labeled first specific binding partner;
(4) bringing the first and second opposable components into operable contact to apply the wash liquid to the sample application zone;
(5) allowing the wash liquid to move through at least a portion of the chromatographic medium including the detection zone; and
(6) observing or measuring the labeled second specific binding partner for the analyte at the detection zone in order to detect or determine the analyte.
Another aspect of the present invention is a multiplex device capable of performing more than one assay in the same device. The device comprises:
(1) a first opposable component including:
(a) at least two sample application zones, one for each assay that can be performed in the device, each sample application zone containing a matrix of porous material permeable to the liquid portion of blood but capable of trapping the cellular components of blood, the sample application zones being laterally spaced on the first opposable component; and
(b) at least two chromatographic media, one for each sample application zone, each chromatographic medium having first and second ends and including:
(i) a detection zone having immobilized thereon a first specific binding partner for the analyte; and
(ii) a conjugate zone having a labeled second specific binding partner for the analyte in resolubilizable form, each sample application zone being in operable contact with the first end of one of the chromatographic media and each conjugate zone being located closer to the first end of each chromatographic medium than each detection zone is located; and
(1) a second opposable component including:
(a) at least two applicators, one for each chromatographic medium; and
(b) at least two absorbers, one for each chromatographic medium.
In the multiplex version of the device, the first and second opposable components are brought into operable contact to cause each applicator to come into operable contact with the corresponding sample application zone to apply a wash liquid thereto and cause each absorber to come into operable contact with the second end of the corresponding chromatographic medium.
Each of the immobilized specific binding partners on the chromatographic media can bind the same analyte. In this alternative, the multiplex assay device performs a number of tests for the same analyte. Alternatively, each of the immobilized specific binding partners on the chromatographic media can bind a different analyte. In this alternative, the multiplex assay device performs tests for a number of different analytes.
Typically, each of the labels of the labeled specific binding powers is the same.
Preferably, the device further comprises at least two barriers to prevent fluid flow between each second applicator and each sample application zone, a barrier being located between each second applicator and each sample application zone.
Preferably, each matrix contains a detergent and a chelating agent, as described above.
Each chromatographic medium can further include a control zone separate from the detection zone. Preferably, each control zone includes analyte immobilized thereto, the analyte being the same analyte as bound by the immobilized first specific binding partner on each chromatographic medium.
Another aspect of the present invention is a test kit incorporating the multiplex assay device of the present invention. The test kit comprises, packaged in separate containers:
(1) the multiplex chromatographic assay device described above; and
(2) a wash liquid for application to each second applicator of the assay device.
Another aspect of the present invention is a method for detecting or determining at least one analyte in at least one test sample comprising the steps of:
(1) applying a test sample to at least one of the sample application zones of the multiplex chromatographic assay device described above;
(2) applying a wash liquid to at least one of the applicators of the chromatographic assay device;
(3) allowing the sample to migrate from the sample application zone through the corresponding chromatographic medium including the conjugate zone and the detection zone to resolubilize the labeled first specific binding partner;
(4) bringing the first and second opposable components into operable contact to apply the wash liquid to the sample application zone;
(5allowing the wash liquid to move through at least a portion of the corresponding chromatographic medium including the detection zone; and
(6observing or measuring the labeled second specific binding partner for the at least one analyte at the detection zone in order to detect or determine the analyte.
Another embodiment of an assay device according to the present invention employs additional specific binding partners to prevent or detect possible interference by a human anti-murine antibody (HAMA) response occurring in a test subject. One of these specific binding partners acts to scavenge antibodies in the test sample that would otherwise bind to the immobilized specific binding partner for the analyte or the mobile labeled specific binding partner for the analyte.
One version of this embodiment comprises:
(1) a first opposable component including:
(a) a sample application zone containing a matrix of porous material permeable to the liquid portion of blood but capable of trapping the cellular components of blood; and
(b) a chromatographic medium having first and second ends and including:
(i) a detection zone having immobilized thereon a first specific binding partner, the first specific binding partner binding the analyte;
(ii) a conjugate zone having a labeled second specific binding partner in a resolubilizable form, the second specific binding partner binding the analyte;
(iii) a capture zone having thereon a third specific binding partner, the third specific binding partner binding human antibodies that bind the first or second specific binding partners; and;
(iv) a validation zone having immobilized thereon a fourth specific binding partner, the fourth specific binding partner binding human antibodies that bind the first or second specific binding partners;
(2) a second opposable component including:
(a) an applicator; and
(b) an absorber.
In this embodiment, the sample application zone is in operable contact with the first end of the chromatographic medium, the conjugate zone is located closer to the first end of the chromatographic medium than the detection zone, the capture zone is located closer to the first end of the chromatographic medium than the conjugate zone, and the validation zone is located closer to the second end of the chromatographic medium than the detection zone.
The first and second opposable components are brought into operable contact to cause the applicator to come into operable contact with the sample application zone to apply a wash liquid thereto and cause the absorber to come into operable contact with the second end of the chromatographic medium.
Another version of this embodiment comprises:
(1) a first opposable component including:
(a) a sample application zone containing a matrix of porous material permeable to the liquid portion of blood but capable of trapping the cellular components of blood, the sample application zone further containing a first specific binding partner; and
(b) a chromatographic medium having first and second ends and including:
(i) a detection zone having immobilized thereon a second specific binding partner, the second specific binding partner specifically binding the analyte;
(ii) a conjugate zone having a labeled third specific binding partner in a resolubilizable form, the third specific binding partner specifically binding the analyte; and;
(iii) a validation zone having immobilized thereon a fourth specific binding partner, the fourth specific binding partner binding human antibodies that bind the second or third specific binding partners;
(1) a second opposable component including:
(a) an applicator; and
(b) an absorber.
In this version of the embodiment, the sample application zone is in operable contact with the first end of the chromatographic medium, the conjugate zone is located closer to the first end of the chromatographic medium than the detection zone, and the validation zone is located closer to the second end of the chromatographic medium than the detection zone. The first specific binding partner at the sample application zone binds human antibodies that bind the second or third specific binding partners. The first and second opposable components are brought into operable contact to cause the applicator to come into operable contact with the sample application zone to apply a wash liquid thereto and cause the absorber to come into operable contact with the second end of the chromatographic medium.
A third version of this embodiment of the present invention comprises:
(1) a first opposable component including:
(a) a sample application zone containing a matrix of porous material permeable to the liquid portion of blood but capable of trapping the cellular components of blood;
(b) a conductor containing a first specific binding partner; and
(c) a chromatographic medium having first and second ends and including:
(i) a detection zone having immobilized thereon a second specific binding partner, the second specific binding partner binding the analyte;
(ii) a conjugate zone having a labeled third specific binding partner in a resolubilizable form, the third specific binding partner binding the analyte; and
(iii) a validation zone having immobilized thereon a fourth specific binding partner that binds human antibodies that bind the second or third specific binding partners;
(1) a second opposable component including:
(a) an applicator; and
(b) an absorber.
In this embodiment, the first specific binding partner binds human antibodies that bind the second or third specific binding partners. The sample application zone is in operable contact with the conductor and the conductor is in operable contact with the first end of the chromatographic medium. The conjugate zone is located closer to the first end of the chromatographic medium than the detection zone, and the validation zone is located closer to the second end of the chromatographic medium than the detection zone. The first and second opposable components are brought into operable contact to cause the applicator to come into operable contact with the sample application zone to apply a wash liquid thereto and cause the absorber to come into operable contact with the second end of the chromatographic medium.
The present invention also encompasses test kits incorporating these versions of this embodiment and assay methods employing these versions.